Method of identifying cytosine methylation patterns in genomic DNA samples

ABSTRACT

The invention concerns a method for the identification of cytosine methylation patterns in genomic DNA samples, wherein
         a) a genomic DNA sample is chemically treated such that cytosine and 5-methylcytosine react differently and a different base pairing behavior of the two products results in the duplex;   b) parts of the thus-treated DNA sample are enzymatically amplified;   c) the amplified parts of the thus-treated DNA sample bind to a surface;   d) a set of probes of different nucleobase sequences, each of which contains the dinucleotide sequence 5′-CpG-3′ at least once, is hybridized to the immobilized DNA sample;   e) the non-hybridized probes are separated;   f) the hybridized probes are analyzed in a mass spectrometer, wherein the position of the probes on the sample holder permits a classification of the hybridizing DNA sample;   g) Assignment of the peak pattern obtained from the mass spectra to the methylation pattern and comparison of the new data with a database.

The invention concerns a method for the identification of cytosinemethylation patterns in genomic DNA samples.

The genetic information which is obtained by complete sequencing ofgenomic DNA as the base sequence only incompletely describes the genomeof a cell. 5-Methylcytosine nucleobases, which are formed by reversiblemethylation of DNA in the cell, are an epigenetic information carrierand serve, for example, for the regulation of promoters. The methylationstate of a genome represents the present status of gene expression,similar to an mRNA expression pattern.

5-Methylcytosine is the most frequent covalently modified base in theDNA of eukaryotic cells. For example, it plays a role in the regulationof transcription, genomic imprinting and in tumorigenesis. Theidentification of 5-methylcytosine as a component of genetic informationis thus of considerable interest. 5-Methylcytosine positions, however,cannot be identified by sequencing, since 5-methylcytosine has the samebase pairing behavior as cytosine. In addition, in a PCR amplification,the epigenetic information that is carried by 5-methylcytosine iscompletely lost.

Several methods are known that attempt to solve these problems. For themost part, a chemical reaction or enzymatic treatment of the genomic DNAis conducted, following which cytosine bases can be distinguished frommethylcytosine bases. A current method is the reaction of genomic DNAwith bisulfite, which leads to a conversion of cytosine bases to uracilin two steps after alkaline hydrolysis (Shapiro, R., Cohen, B., Servis,R. Nature 227, 1047 (1970). 5-Methylcytosine remains unchanged underthese conditions. The conversion of C to U leads to a modification ofthe base sequence, from which the original 5-methylcytosines can now bedetermined by sequencing (only these still supply a band in the C lane).

A review of other known possibilities for detecting 5-methylcytosine canbe derived, for example, from the following review article: Rein, T.,DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 26, 2255 (1998).

With a few exceptions (e.g., Zeschnigk, M. et al., Eur. J. Hum. Gen. 5,94–98; Kubota T. et al., Nat. Genet. 16, 16–17), the bisulfate techniquehas been used previously only in research. Short, specific pieces of aknown gene are always amplified, however, after a bisulfite treatmentand either completely sequenced (Olek, A. and Walter, J., Nat. Genet.17, 275–276) or individual cytosine positions are detected by a“primer-extension reaction” (Gonzalgo, M. L. and Jones. P. A., Nucl.Acids Res. 25, 2529–2531) or by enzyme cleavage (Xiong, Z. and Laird, P.W., Nucl., Acids Res. 25, 2532–2534). All of these references derivefrom the year 1997. The concept of using complex methylation patternsfor correlation with phenotypic data of complex genetic disorders isonly mentioned in DE-195 43065 A1. For example, the actual detection isnot conducted herein by analysis of the hybridization of nucleic acidsamples in the mass spectrometer.

It is not always necessary to actually determine the entire sequence ofa gene or gene segment, as is the goal in the case of sequencing. Thisis particularly the case if a few 5-methylcytosine positions within along base sequence are to be scanned for a multiple number of differentsamples. Here sequencing supplies redundant information to a greatextent and is also very expensive. This is also the case if the sequenceis already known and methylation positions exclusively are to be found.It is also conceivable that in several cases, only the differences inthe methylation pattern between various genomic DNA samples are ofinterest in general and that the determination of a multiple number ofthe same methylated positions can be dispensed with. For the questionsintroduced here, up until now, there has existed no method whichsupplies the desired results in a cost-favorable manner withoutsequencing each individual sample.

Sequence information also needs to be determined less often, since thegenome project, whose goal is the complete sequence of variousorganisms, is rapidly progressing. In fact, at the present time,approximately 5% of the human genome has been sequenced completely, butnow, since other genome projects are concluding and sequencing resourcesare made available in this way, another 5% is added every year. Thecomplete sequencing of the human genome is expected by the year 2006.

Matrix-assisted laser desorption/ionization mass spectrometery (MALDI)is a new, very powerful development for the analysis of biomolecules(Karas, M. and Hillenkamp, F. 1988. Laser desorption ionization ofproteins with molecular masses exceeding 10,000 Daltons. Anal. Chem. 60:2299–2301). An analyte molecule is embedded in a matrix absorbing in theUV. The matrix is evaporated in vacuum by a short laser pulse and theanalyte is transported unfragmented into the gas phase. An appliedvoltage accelerates the ions in a field-free flight tube. Ions areaccelerated to varying degree on the basis of their different masses.Smaller ions reach the detector sooner than larger ions. Thetime-of-flight is converted to the mass of the ions.

Technical innovations of the hardware have significantly improved themethod. The “delayed extraction” (DE) method is worthy of mention. ForDE, the acceleration voltage for the laser pulse is turned on with adelay, and in this way an improved resolution of signals is achieved,since the number of collisions is reduced.

MALDI is excellently suitable for the analysis of peptides and proteins.For nucleic acids, the sensitivity is approximately 100 times poorerthan for peptides and decreases overproportionally with increasingfragment size. The reason for this lies in the fact that only a singleproton must be captured for the ionization of peptides and proteins. Fornucleic acids, which have a backbone with a multiple negative charge,the ionization process via the matrix is essentially inefficient. ForMALDI, the choice of matrix plays an extremely important role. For thedesorption of peptides, several very powerful matrices have been found,which result in a very fine crystallization. In fact, several suitablematrices have now been found for DNA, but the difference in sensitivitywas not reduced thereby. Phosphorothionate nucleic acids, in which theusual phosphates of the backbone are substituted by thiophosphates, canbe converted to a charge-neutral DNA by simple alkylation chemistry.

The coupling of a “charge tag” to this modified DNA results in anincrease of sensitivity to the same range as is found for peptides. Bythese modifications, it is now also possible to utilize matrices thatare similar to those that are used for the desorption of peptides.Another advantage of charge tagging is the increased stability ofanalysis when impurities are present, which greatly complicate thedetection of unmodified substrates. PNAs and methylphosphonateoligonucleotides have been investigated with MALDI and can thus beanalyzed.

Presently this technology can distinguish molecules with a massdifference of 1 Da in the mass region from 1,000 to 4,000 Da. Due to thenatural distribution of isotopes, most biomolecules, however, areapproximated within a range of 5 Da. Technically, thismass-spectrometric method is thus very suitable for the analysis ofbiomolecules. Reasonably, the products to be analyzed, which are to bedistinguished, must lie at least 5 Da apart from one another. Therefore,600 molecules could be distinguished in this mass region.

An array with many thousand target DNAs can be immobilized on asolid-phase support and then all of these target DNAs can beinvestigated jointly for the presence of a sequence by means of a probe(nucleic acid with complementary sequence).

A correspondence of the target DNA with the probe is achieved by ahybridization of the two parts with one another. Probes can be anynucleic acid sequences of any length. Different methods exist for theselection of optimal libraries of probe sequences, which minimallyoverlap. Probe sequences can be prepared for the purpose of findingspecific target DNA sequences. Oligofingerprinting is an approach inwhich this technology is utilized. A library of target DNAs is scannedwith short nucleic acid probes. For the most part, the probes in thiscase are only 8–12 bases long. Each time a probe is hybridized once ontoa target DNA library immobilized on a nylon membrane. The probe isradioactively labeled and the hybridization is evaluated on the basis oflocalizing the radioactivity. For scanning an immobilized DNA array,fluorescently labeled probes have also been used.

U.S. Pat. No. 5,605,798 describes the scanning of target nucleic acidsthat have been immobilized by hybridizing with nucleic acid probes andmass spectrometry. However, an identification of methylation patterns isnot specifically conducted, nor are modified nucleic acids (e.g. PNAs,charge tags) utilized, nor is a genome amplification conducted.

Any molecules can be used as probes, which can interact in asequence-specific manner with a target DNA. Oligodeoxyribonucleotidesare used most often currently. However, any modification of nucleicacids, e.g., peptide nucleic acids (PNA), phosphorothioateoligonucleotides or methylphosphonate oligonucleotides can be used. Thespecificity of a probe is most essential. Phosphorothioateoligonucleotides are not entirely suitable, since their structure isdisrupted by sulfur atoms and the hybridization property is alsodisrupted thereby. A reason for this could be that the phosphorothioateoligonucleotides are normally not synthesized as pure diastereomers. Inthe case of methylphosphonate oligonucleotides, a similar problemexists, but these oligonucleotides are synthesized and propagated aspure diastereomers. An essential difference in this modification is theuncharged backbone, which leads to a reduced dependence of hybridizationon buffer salts and overall leads to higher affinity due to fewerrepulsions. Peptide nucleic acids also have an uncharged backbone, whichsimultaneously deviates chemically very greatly from the familiarsugar-phosphate structure of the backbone in nucleic acids. The backboneof a PNA has an amide sequence instead of the sugar-phosphate backboneof conventional DNA. PNA hybridizes very well with DNA of complementarysequence. The melting point of a PNA/DNA hybrid is higher than that ofthe corresponding DNA/DNA hybrid and again the dependence ofhybridization on buffer salts is relatively small.

Combinatory syntheses, i.e., the preparation of substance librariesproceeding from a mixture of precursors, are conducted both in the solidphase as well as in the liquid phase. Combinatory solid-phase synthesiscan be completed in a very short time, since in this case, theseparation of byproducts is very simple. Only the target compounds thatare bound to the support are retained in one washing step and areisolated at the end of the synthesis by the targeted cleavage of alinker. This technique permits in a simple way the simultaneoussynthesis of a multiple number of different compounds on a solid phaseand thus obtaining chemically “pure” substance libraries.

Compound classes, which are also synthesized on a solid phase innon-combinatory, conventional syntheses, are thus particularlyaccessible to combinatory chemistry and are consequently also verywidely used. This particularly concerns peptide, nucleic acid and PNAlibraries.

Peptides are synthesized by binding the first N-protected amino acid(e.g., Boc) to the support, subsequent de-protection and reaction of thesecond amino acid with the NH₂ group that has been released from thefirst. Unreacted amino functions are withdrawn in an additional“capping” step of a further reaction in the next synthesis cycle. Theprotective group of the amino function of the second amino acid isremoved and the next building block can be coupled. A mixture of aminoacids is used in one or more steps for the synthesis of peptidelibraries. The synthesis of PNA and PNA libraries is conductedrationally.

Nucleic acid libraries are obtained for the most part by solid-phasesynthesis with mixtures of different phosphoramidite nucleosides. Thiscan be carried out on commercially obtainable DNA synthesizers withoutmodifications in the synthesis protocols.

Various studies for combinatory synthesis of PNA libraries have beenpublished. These studies concern the structure of combinatory sequences,i.e., the synthesis of PNAs in which individual, specific bases in thesequence are replaced by degenerated bases and in this way randomsequence variance is achieved.

The use of mass-spectrometric methods for the analysis of combinatorylibraries has been described many times.

Different methods exist for immobilizing DNA. The best known method isthe fixed binding of DNA, which is functionalized with biotin, to astreptavidin-coated surface. The binding strength of this systemcorresponds to a covalent chemical bond without being one. In order tobe able to bind a target DNA covalently to a chemically preparedsurface, a corresponding functionality of the target DNA is required.DNA itself does not possess a functionalization that is suitable. Thereare different variants in a target DNA for introducing a suitablefunctionalization: two easy-to-manipulate functionalizations areprimary, aliphatic amines and thiols. Such amines are quantitativelyconverted with N-hydroxy succinimide esters, and thiols reactquantitatively with alkyl iodides under suitable conditions. However, itis difficult to introduce such a functionalization into a DNA. Thesimplest variant is introduction by means of a primer of a PCR. Targetedvariants utilize 5′-modified primers (NH₂ and SH) and a bifunctionallinker.

An essential component for immobilization onto a surface is the natureof this surface. Systems described up until now are primarily comprisedof silicon or metal (magnetic beads). Another method for binding atarget DNA is based on using a short recognition sequence (e.g., 20bases) in the target DNA for hybridizing to a surface-immobilizedoligonucleotide.

Enzymatic variants have also been described for introducing chemicallyactivated positions into a target DNA. Here, a 5′-NH₂ functionalizationis enzymatically introduced in a target DNA.

The object of the present invention is to create a method, whichovercomes the disadvantages of the state of the art and can indicatecytosine methylations effectively and in a highly parallel manner, in anarray of immobilized genomic DNA samples.

The subject of the present invention is thus a method for findingepigenetic information carriers in the form of 5-methylcytosine bases ingenomic DNA, which uses a multiple number of probes simultaneously formass-spectrometric investigation of an array of target nucleic acids.

The object is solved according to the invention by making available amethod for the identification of cytosine methylation patterns ingenomic DNA samples, by:

-   -   a) chemically treating a genomic DNA sample in such a way that        cytosine and 5-methylcytosine react differently and obtaining a        different base pairing behavior of the two products in the        duplex;    -   b) enzymatically amplifying portions of the thus-treated DNA        sample;    -   c) binding the amplified portions of the thus-treated DNA sample        to a surface;    -   d) hybridizing a set of probes of different nucleobase        sequences, each of which contains the dinucleotide sequence        5′-CpG-3′ at least once, to the immobilized DNA sample;    -   e) separating the non-hybridized probes;    -   f) analyzing the hybridized probes in a mass spectrometer,        wherein the position of the probes on the sample holder permits        a classification of the hybridizing DNA sample;    -   g) assignment of the peak pattern obtained from the mass spectra        to the methylation pattern and comparison of the new data with a        database.

It is preferred according to the invention that one or more amplifiedgenomic DNA fragments is (are) immobilized in c) by hybridization withcomplementary oligonucleotide or PNA sequences, which are boundcovalently to the surface.

It is further preferred according to the invention that after thehybridization, a cross-linking of the genomic DNA fragments is producedwith the oligonucleotide or PNA sequences bound to the surface. It isparticularly preferred here that covalent chemical bonds are formed forthe cross-linking. It is also preferred according to the invention thatelectrostatic interactions are formed for the cross-linking.

It is of further advantage that the oligonucleotide or PNA sequenceswhich are bound to the surface contain 5-bromouracil structural units.

It is preferred according to the invention that the immobilizedcomplementary oligonucleotide sequences contain modified bases, riboseor backbone units.

The method according to the invention is further characterized in thatthe genomic DNA probe is propagated in b) in the form of severalamplified fragments, so that at least 0.01% of the entire genome isamplified.

It is also preferred according to the invention that the mixture ofamplified DNA fragments is bound onto a surface on which a multiplenumber of different points is arranged, each of which can bind differentportions of the amplified DNA sample.

According to the invention, it is further preferred that a set of probesbe used in d), which contains the dinucleotide sequence 5′-CpG-3′ onlyonce per probe and otherwise, each of the probes contain either nocytosine or no guanine bases.

It is also preferred according to the invention that a bisulfite orpyrosulfite or disulfite solution or a mixture of the indicatedsolutions is used in step a), together with other reagents, for thespecific or sufficiently selective conversion of cytosine to uracil.

It is also advantageous that the surface used for the immobilization ofamplified sample DNA is also the sample holder for a mass spectrometer.It is preferred that the surface used for the immobilization ofamplified sample DNA is introduced as a whole, prior to f), onto asample holder for a mass spectrometer. It is also preferred here thatthe hybridized probes are stripped from the immobilized, amplified DNAsamples before, after, or by contact with a matrix.

It is further preferred according to the invention that the probes arenucleic acids, which bear one or several mass tags. It is alsoadvantageous according to the invention that one or more mass tags arealso charge tags. Or that the probes also have a charge tag.

It is preferred according to the invention that the probes are modifiednucleic acid molecules. It is particularly preferred that the modifiednucleic acid molecules are PNAs, alkylated phosphorothioate nucleicacids or alkyl phosphonate nucleic acids.

It is preferred according to the invention that the probes are producedby combinatory synthesis. It is particularly preferred according to theinvention that the various base structural units are labeled in such away that each of the probes synthesized from them can be distinguishedby their mass in the mass spectrometer.

It is of further advantage according to the invention that the probesare produced as sublibraries and are provided with various mass and/orcharge tags.

It is most preferred according to the invention that matrix-assistedlaser desorption/ionization mass spectrometry (MALDI) is used in f).

Another subject of the present invention is a kit for conducting themethod according to the invention, which contains the following: asample holder for a mass spectrometer, which is modified such thatrandomly selectable parts of a genome can be immobilized onto thisholder, and/or probe libraries with which the DNA immobilized to thesample holder is analyzed by the mass spectrometer, and/or otherchemicals, solvents and/or adjuvants, as well as, optionally,instructions for use.

The method according to the invention serves for the identification of5-methylcytosine positions in genomic DNA which can have variousorigins. The genomic DNA is first treated chemically in such a way thatthere is a difference in the reaction of cytosine bases and5-methylcytosine bases. Possible reagents here are, e.g., disulfite(also designated bisulfite), hydrazine and permanganate. In a preferredvariant of the method, the genomic DNA is treated with disulfite in thepresence of hydroquinone or hydroquinone derivatives, whereby thecytosine bases are selectively converted into uracil after subsequentalkali hydrolysis. 5-Methylcytosine remains unchanged under theseconditions. After a purification process, which serves for theseparation of excess disulfite, specific segments of the pretreatedgenomic DNA are amplified in a polymerase reaction. In a preferredvariant of the method, the polymerase chain reaction is used here. In aparticularly preferred variant of the method, the polymerase chainreaction is conducted in such a way that at least 0.01% of the totalgenome is amplified in the form of several fragments.

The amplified, pretreated DNA sample can now be immobilized onto asurface in several variants of the method. In a preferred variant of themethod, immobilization to the surface is conducted in such a way thatthe surface has been modified beforehand with oligonucleotides or shortPNA (peptide nucleic acid) sequences and thus a hybridization ofcomplementary sequences in the DNA sample results. Basically, theimmobilized oligonucleotides can be modified at bases, at (deoxy)riboseand/or at the backbone, in contrast to conventional DNA. Now ifdifferent oligonucleotide or PNA sequences are bound to this surface inthe form of an array or are synthesized on it, each of these differentsequences can bind different portions of the amplified DNA fragments. Ina particularly preferred variant of the method, a cross-linking of thetwo strands is conducted subsequent to the hybridization. This canresult from the formation of a covalent chemical bond or a stableelectrostatic interaction. In another preferred variant, a photochemicalcross-linking is conducted by means of bromouracil structural units.

It is also possible to separately amplify fragments of the pretreatedgenomic DNA and to immobilize the products individually at differentsites on the surface. In a preferred variant of the method, this isperformed in such a way that one of the PCR primers bears a functionsuitable for immobilization, which can enter into a bond with afunctionality introduced onto the surface.

The surface, to which the amplified DNA fragments are bound, will eitherbe transferred onto the sample holder of a MALDI mass spectrometer orwill be this sample holder itself. The construction and software of themass spectrometer thus assure that the investigated point on the sampleholder can be assigned each time to the sample originally bound there.

A set of probes is now hybridized to the immobilized, amplified DNAfragments, whereby these probes each contain the sequence 5′-CpG-3′ atleast once and otherwise do not contain either cytosine or guaninebases. The probes can be oligonucleotides, modified oligonucleotides orPNAs (peptide nucleic acids). In a preferred variant of the method, thisset of probes is produced as a combinatory library in a combinatorysynthesis approach. In another preferred variant of the method, theprobes can be clearly distinguished by their mass, so that it ispossible to conclude the sequence from the mass. For this purpose, theprobes can be provided with mass tags, which prevent the various probesfrom being of equal mass. The probes can be provided with charge tags inorder to achieve a better presentation in the mass spectrometer and toincrease the the analysis in the presence of salts and detergents. Themass tags may also be charge tags. The probes may also be prepared ascombinatory sublibraries, which in turn bear different mass and/orcharge tags. The probes can be PNAs, unmodified nucleic acid moleculesor modified nucleic acid molecules such as phosphothioate nucleic acids,alkylated phosphorothioate nucleic acids or alkyl phosphonate nucleicacids, regardless of further modification by mass and charge tags.

The non-hybridized probes are separated in one or more washing steps.The hybridized probes thus remain at their positions.

The surface is fastened to the MALDI sample holder and transferred tothe mass spectrometer or transferred directly if the method has beenconducted on the MALDI sample holder itself. The array of samples is nowinvestigated by mass spectrometer on hybridized probes. The hybridizedprobes are dehybridized for this purpose by contact with the MALDImatrix and embedded in it in a preferred variant of the method; howeverno cross-contamination of adjacent points results due to the rate atwhich the matrix is introduced. The hybridized probes provide a peakpattern at each point, by means of which the sequence can be derived, atwhich a hybridization has occurred. Due to the pretreatment (preferablywith bisulfite), different sequences result for DNA fragments methylateddifferently at the cytosine. Therefore, each of the characteristicmethylation patterns of the investigated DNA sample is the peak patternproduced by the probe in the mass spectrometer. Then these methylationpatterns are compared with those of a database.

1. Method for identifying cytosine methylation patterns in genomic DNAsamples, said method comprising the steps of: a) chemically treating agenomic DNA sample in such a way that cytosine and 5-methylcytosinereact differently and a different base pairing behavior of the twoproducts is obtained in the duplex; b) enzymatically amplifying portionsof the thus-treated DNA sample; c) binding the amplified portions of thethus-treated DNA sample to a surface; d) contacting a set of probes ofdifferent nucleobase sequences, each of which contains the dinucleotidesequence 5′-CpG-3′ at least once, to the immobilized DNA samples forhybridization to distinguish methylated and nonmethylated cytosines insaid genomic DNA sample; e) removing any non-hybridized probes from theimmobilized DNA samples; f) analyzing the hybridized probes in a massspectrometer, wherein the position of the hybridized probes on thesurface permits a classification of the immobilized DNA samplehybridized thereto; g) assigning a peak pattern obtained from the massspectra to a methylation pattern for the immobilized DNA and comparingthe peak pattern with a database to identify cytosine methylationpatterns in the genomic DNA sample.
 2. Method according to claim 1,further characterized in that one or more amplified genomic DNAfragments are immobilized in c) by hybridization with complementaryoligonucleotide or PNA sequences, which are covalently bound to thesurface.
 3. Method according to claim 2, further characterized in that across-linking of the genomic DNA fragments with the oligonucleotide orPNA sequences bound to the surface results after the hybridization. 4.Method according to claim 3, further characterized in that covalentchemical bonds are formed for the cross-linking.
 5. Method according toclaim 3, further characterized in that electrostatic interactions areformed for the cross-linking.
 6. Method according to claim 3, furthercharacterized in that the oligonucleotide or PNA sequences bound to thesurface contain 5-bromouracil structural units.
 7. Method according toclaim 1, further characterized in that the immobilized complementaryoligonucleotide sequences contain modified bases, ribose or backboneunits.
 8. Method according to claim 1, further characterized in that thegenomic DNA sample is propagated in b) in the form of several amplifiedfragments, so that at least 0.01% of the total genome is amplified. 9.Method according to claim 1, further characterized in that the mixtureof amplified DNA fragments is bound to a surface, on which a multiplenumber of different points is arranged, each of which can bind differentportions of the amplified DNA sample.
 10. Method according to claim 1,further characterized in that a set of probes is used in d), whichcontains the dinucleotide sequence 5′-CpG-3′ only once in each probe andthe probes otherwise contain either no cytosine or no guanine bases. 11.Method according to claim 1, further characterized in that a bisulfiteor pyrosulfite or disulfite solution or a mixture of the indicatedsolutions is used together with other reagents for the specific orsufficiently selective conversion of cytosine to uracil.
 12. Methodaccording to claim 1, further characterized in that the surface used forthe immobilization of amplified sample DNA is also the sample holder fora mass spectrometer.
 13. Method according to claim 1, furthercharacterized in that the surface used for the immobilization ofamplified sample DNA is introduced as a whole, prior to f), onto asample holder for a mass spectrometer.
 14. Method according to claim 1,further characterized in that the hybridized probes are stripped fromthe immobilized amplified DNA samples before, after or by contact with amatrix.
 15. Method according to claim 1, further characterized in thatthe probes are nucleic acids, which bear one or more mass tags. 16.Method according to claim 15, further characterized in that one or moremass tags are also charge tags.
 17. Method according to claim 15,further characterized in that the probes also bear a charge tag. 18.Method according to claim 1, further characterized in that the probesare modified nucleic acid molecules.
 19. Method according to claim 2,further characterized in that nucleic acid molecules are selected fromthe group consisting of PNAs, alkylated phosphorothioate nucleic acidsand alkyl phosphonate nucleic acids.
 20. Method according to claim 1,further characterized in that the probes are prepared by combinatorysynthesis.
 21. Method according to claim 20, further characterized inthat different base structural units are labeled in such a way that eachof the probes synthesized from them can be distinguished by their massin the mass spectrometer.
 22. Method according to claim 1, furthercharacterized in that the probes are prepared as sublibraries and theseare provided with different mass and/or charge tags.
 23. Methodaccording to one of the preceding claims, further characterized in thatmatrix-assisted laser desorption/ionization mass spectrometry (MALDI) isconducted in f).